We have previously shown that the frequency of the type 4 allele of apolipoprotein E (APOE4) is significantly increased in late-onset familial and sporadic AD. APOE4 acts in an autosomal dose-dependent manner to increase the risk and decrease the mean age of onset of AD. Both amyloid beta-peptide (Abeta) and tau bind to the apoE protein with high specificity and avidity. The interaction with both proteins is apoE isoform-specific over a range of concentrations. ApoE does not appear to be synthesized is neurons; however, apoE immunoreactivity is present in the hippocampal neurons in 24/24 patients with AD. 12/13 patients with Parkinson's disease (PD) or AD/PD, and 2/6 non-demented controls. These data suggest that apoE may be involved in intraneuronal metabolism. We would like to test the hypothesis that isoform-specific, differential intracellular processing of exogenous apoE isoforms occurs using the well characterized cell system of human fibroblasts and standard in vitro cell biological techniques. The basic biology of exogenous apoE uptake will be examined in cells derived from patients with familial late-onset AD and two forms of familial early- onset AD, one linked to chromosome 14 and the other carrying the APP717val- ile mutation, to determine if there are detectable APOE genotype-associated differences. We plan to use exogenously added 125I-labeled apoE isoforms to follow the binding, internalization, and ultimate subcellular fate using pulse-chase protocols followed by subcellular fractionation. Parallel pulse-chase experiments using ferritin-tagged apoE isoforms and electron microscopy are also planned. We further propose to determine the localization of endogenous apoE in subcellular fractions of genetically determined, AD-affected fibroblasts using Western blot analyses. We will also address whether Abeta and other proteins are complexed with intracellular or extracellular endogenous apoE.